This invention relates to an apparatus for processing biological samples on slides for a wide variety of purposes. Biological samples are analyzed for many purposes using a variety of different assays. Pathologists often use histochemistry or immunocytochemistry for analyzing biological samples, molecular biologists may perform in situ hybridization or in situ polymerase chain reactions on biological samples, etc. Often the sample to be analyzed will be embedded in paraffin and mounted on a microscope slide.
The assays usually involve the use of antibodies, enzymes and other expensive reagents and it is desirable to keep reagent volume use to a minimum to lower costs. These assays are also quite labor intensive although there are now some automated systems (e.g., the Ventana ESIHC Staining System, the Shandon Lipshaw Cadenza Automated Immunostainer; also see Brigati et al. (1988)). The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References. Most automated systems can only perform 40 to 48 slides per run. Fisher automated systems can perform 120 slides per run. Most automated systems which only perform immunocytochemistry do not perform deparaffinizing, histochemistry (such as hematoxylin and eosin staining) and coverslipping steps and these consequently must be done separately by hand which is time and labor intensive. The automated systems perform only a small part of the overall process of preparing and analyzing slides. Steps which are still manually performed-prior to the automated portion include sorting of cases and slides, labeling slides, programming the automated equipment, daily antibody and reagent preparation, preparing control tissue which is mounted on slides, and microwave antigen retrieval. Procedures still performed manually after the automated steps are dehydration, coverslipping, slide labeling and sorting of slides and cases. Furthermore, most commercial ready-to-use reagents are not suitable for automated systems which are required to use specially designed reagents. Laboratories which process large numbers of samples are likely to be willing to pay the high cost associated with buying these automated systems as well as the high cost of using the disposable accessories and reagents to perform the assays, but small to intermediate sized laboratories find it more cost effective to continue to process samples manually.
A typical immunocytochemistry assay requires a series of many steps. These include: obtaining a biological sample such as from a biopsy, fixing the sample in formalin, processing the sample overnight, embedding the sample in paraffin, cutting serial sections and mounting on microscope slides and drying. These steps are followed by steps to deparaffinize (treatments in xylene, ethanol and water), and finally the reaction can be performed on the sample which has been mounted on the slide. Typically a series of solutions including reagents such as enzymes, primary antibody, secondary antibody, detection reagent, chromogen, counterstain, etc. is dropped onto the slide, incubated, and washed off. Finally the sample may be viewed under the microscope. Clearly there are many individual steps involved and each sample on a slide must be processed individually. Besides being very labor intensive, there are drawbacks associated with the commonly used method of simply dropping solutions on top of the mounted sample on the microscope slide. The solution is not restricted simply to the area of the biological sample itself and the solution may be relatively deep rather than being a thin layer. These features require use of extra reagents which are quite expensive. Leaving the solutions open to the air as they sit on the slide also may lead to evaporation if the samples must incubate for a long period of time. Evaporation leads to concentration or drying out of the reagents and high concentrations may lead to increased background levels which are clearly undesirable. If the solutions evaporate totally the assay will fail. Incubating samples in humidity chambers with covers may prevent evaporation problems, but water droplets which condense onto the humidity chamber cover may fall onto the slides and this will ruin the assay.
Improved methods for more rapidly assaying several samples at once, but without the high cost of automated systems, will be welcomed by small to intermediate sized laboratories. Furthermore, methods which will allow use of smaller amounts of reagents and overcome the drawbacks of processing samples on slides open to the atmosphere will be a welcome advance.
The present invention is an apparatus for performing manual assays on biological samples mounted on microscope slides. One aspect of the invention is a multislide slideholder capable of holding multiple standard microscope slides, preferably 3-10 slides and more preferably 3 or 6 slides, thereby allowing for the processing of multiple samples at one time. A second aspect of the invention is a multiwell tray containing multiple shallow wells, preferably 3-10 and more preferably 3 or 6 such wells, into which reagents are placed and upon which the slideholder plus slides is placed. A third aspect of the invention is a set of prealigned and prespaced coverslips for rapidly placing said coverslips onto the processed slides. Another aspect of the invention is a second type of multiwell tray which is useful in automating several of the steps of the procedures.
Besides this new design of a slideholder and corresponding tray and coverslips, other aspects of the invention are set out which aid in making assays more rapid and convenient. One such aspect is the use of reagents which are predried in the wells of the tray thereby simply necessitating the addition of water or buffer to the well without having to add the reagents at the time of assay. The well is then covered with a slide with a biological sample premounted on the slide. The different wells of a multiwell tray can be pretreated with different reagents dried in each well. Multistep assays can be performed by moving a slideholder with attached slides from one multiwell tray to the next, with each well of a multiwell tray having the desired reagents predried on it. A variation of this is to employ a multilayer coating of reagents in each well such that the first set of reagents dissolves quickly and acts upon the biological sample, the second layer then dissolves releasing the reagents for the second step, etc., thereby requiring the use of fewer trays, possibly only a single tray.
Another aspect of the invention is to have built in controls on each slide. This is a portion of the slide to which are attached positive and negative controls. These controls allow one to determine whether the assay has worked properly for each individual slide since each slide has its own set of controls and which simultaneously act as labels for each slide.
The slideholder is designed in conjunction with the tray. The purpose of the slideholder is to have multiple slides, preferably up to six slides, all attached to a single holder so that all the attached slides may be processed simultaneously throughout all of the steps of the staining procedure from deparaffinizing to coverslipping without ever separating the slides from the slideholder. This is a labor intensive step and the ability to process multiple slides at once rather than processing slides individually is an important aspect of the invention. Since one technician typically is capable of easily processing about 40-50 individual slides without mistakes, using a slideholder with six slides per slideholder will allow a single technician easily to perform approximately 240-300 slides for routine histochemistry and immunochemistry staining. This is about 2-6 times as many slides as handled by automated systems per each run.
One useful aspect of the present invention is that any kind of commercial ready-to-use reagents are compatible with the slideholder and there is no need to use specially designed reagents. Another useful aspect is that the apparatus allows a technician to perform different staining procedures on the different slides, e.g., histochemistry and immunocytochemistry or in situ hybridization and in situ PCR at the same time. Other important aspects of the present invention are that it allows one to observe chromogen color development as it is happening and it also gives results which have at least as low a background and sometimes a lower background level of staining than is seen in conventional techniques.
Clearly slideholders capable of holding more than six slides may be envisioned. However there are two practical reasons for utilizing holders capable of holding three or six slides. The first such reason is that the staining dishes in which slides are processed, e.g., washing of the slides, which are presently in use in the typical pathology lab are wide enough to handle only up to six slides side by side at a single time. Therefore a design to hold up to six slides will be compatible with presently used equipment. A second reason for designing a system for processing three or six slides at one time is that in a typical pathology assay three samples must be run together, these being the actual sample being assayed plus a known positive control plus a known negative control. Since a single assay typically requires the use of three slides a system capable of handling six slides allows for the processing of two patient samples at a time.
The slideholder may be designed in different ways. The purpose of the slideholder is to hold up to six microscope slides at one time so that one can easily manipulate the six slides simultaneously with one hand. In one embodiment a reusable slideholder is used. The reusable slideholder is designed to allow the tops of microscope slides to fit into the holder simply by inserting the slides into slots in the holder. The slides will fit firmly so as not to fall out during handling. The holder positions the slides in the slots so that the slides are prealigned to fit onto the wells of the tray containing the reagents which will react with the biological samples. The thickness of the holder is designed so that it fits into a trough in the tray and keeps the microscope slides level on top of the wells. If the holder is too thick or too thin the slides may not rest properly on top of the wells in the lower tray and proper contact will not be made with the reagents in the wells.
The slideholder need not be a reusable one. In this second embodiment of the invention one end portion of each microscope slide is glued to a slideholder using a glue which is xylene and ethyl alcohol resistant. The glue holds the slides firmly to the slideholder during processing and when the processing, e.g., staining, is completed the slides can be easily separated from the slideholder.
A third embodiment of the invention is a slideholder with suction cups attached wherein said suction cups hold the slides firmly on the slideholder.
A fourth embodiment of a slideholder is one in which the slide holder consists of two plastic portions with ridges wherein one portion is placed onto each side of the slides and then clipped together such that the slides are held between the two portions of the slideholder. The ridges properly align and space the slides. In one variation of this and other embodiments, the slideholder has ribbed surfaces of plastic or rubber which help to hold the slides firmly in place.
In any embodiment of a slideholder it is useful to have a handle portion to make the handling of the slideholder simpler, but the presence of a handle is not essential. If a handle is present it is useful to have holes in the handle through which a xe2x80x9cforkxe2x80x9d can be inserted such that several (15-20) slideholders can be placed onto a single fork and manipulated together easily. Another useful variation that is applicable to any embodiment of slideholder is to have an opening or a transparent region of the slideholder which acts as a window through which one can see a label attached to the end of the slide which is inserted into the slideholder. Yet another desirable feature which may be used is to have slideholders of various colors which make it simple to determine which slides are undergoing which assay when a variety of assays is being performed.
The tray to contain the reagents such as antibodies and enzymes is an integral part of some embodiments of the invention. The tray is preferably designed with three or six wells although trays with a different number of wells may also be used. In one embodiment of the present invention, each well is shallow to hold a minimal amount of reagent to keep costs low but is deep enough to allow for fluid motion within the well. This overcomes some problems present with systems requiring capillary action of fluid between two slides, especially when viscous reagents must be used. See, Babbitt et al., U.S. Pat. No. 5,002,736; D. J. Brigati, U.S. Pat. No. 4,777,020; Bowman et al., U.S. Pat. No. 4,985,206; and McGrath et al., U.S. Pat. No. 5,192,503. Each well is completely separated from neighboring wells by a trough so that any overflow from one well cannot contaminate a neighboring well. A tray may be designed without these intervening troughs if one is not concerned about contamination between wells, e.g., if all of the wells contain the same solution. The microscope slides with mounted biological samples are placed sample side down on top of the wells of the tray and completely cover the wells effectively sealing the wells from the atmosphere. This aspect of the invention prevents evaporation of the small amount of fluid in the well. It further prevents contamination of the reagents in the well and also overcomes the problem of extraneous matter falling on top of the sample such as sometimes occurs when samples are incubated in a covered humidity chamber and drops of water fall onto the surface of the slides. In the present invention any such drops of water fall onto the backs of the incubating slides since the slides are placed sample side down onto the trays.
Another embodiment of the tray is one in which the bottom of each well is made of a soft or pliable material. One advantage to having a soft bottom is that it becomes easy to remove air bubbles which may be trapped between the slide and soft bottom. By pushing on the soft bottom of a well, one can easily remove air bubbles to a region away from the region of the biological sample. A second advantage to having a soft bottom is that the volume of reagent solution needed in a well becomes flexible.
A related embodiment is the use of a tray with a flexible bottom. The bottom may be soft or it may be a hard material which is capable of being moved. This movement can be as simple as applying pressure to a solid plastic tray bottom to make it flex or it can be more complex such as a hinged bottom. The purpose of such a flexible bottom is that the volume within a well can be adjusted. This is useful because if one is performing a reaction, e.g., PCR, in a very small volume of about 10-15 xcexcL in the well, it is very difficult to pump this small volume out of the well because of the force of the capillary action of the small amount of liquid between the tray and the covering slide with biological sample. To overcome this, the well volume can be made small to encompass only the 10-15 xcexcL volume when desired following which the volume can be increased to accommodate more fluid so that the fluid can be easily pumped through the well and collected if desired.
Another feature of the tray is two notches or channels (which act as ports or vents) in the well boundaries and tubing attached to the outside of the boundary at one of these channels. These features allow the slide holder plus slides to be placed onto the tray prior to adding reagents to the tray. The reagent solutions may be added through the tubing. A simple way of adding solutions between the slides and the wells of the tray is to immerse the tray with slideholder and slides vertically into the solutions. Expensive reagents can be pipetted directly through a notch or port. The solutions pass from the tubing through a first channel or port or while air in the well escapes through a second channel (a vent) at the top of the boundary.
Yet another possible feature of a tray is a channel through which reagents can be added. Such a channel can be centrally located as shown by feature 430 in FIG. 16B. Such a channel can extend from the absolute base of the tray through the base and into a well region of the tray. Effectively this means that each well has a hole in its bottom. If desired a second channel or hole can be included in the bottom of each well to act as a vent for air to escape as reagent is added through the first channel.
Other alternate embodiments of the tray may be used but will not necessarily have all of the advantages outlined above. One such alternate embodiment is to design a tray with larger wells such that each well can accommodate multiple slides at one time, preferably 3 or 6. In this design slides are placed into a holder which holds the slides in tight conjunction side by side. In a design with a tray containing wells large enough to require 3 slides to completely cover each well, a slideholder will hold 3 slides in tight conjunction (or 2 sets of 3 slides with a space between the 2 sets). With this configuration it is necessary to make only a single pipetting to fill a well for 3 slides rather than requiring 3 individual pipetting steps for the 3 slides. This is a labor saving advantage. The disadvantage is that the volume of a single well to be covered by 3 slides is greater than 3 times the volume of a well designed to be covered by an individual slide. This is because wells designed for single slides are narrower than the full width of a slide. This alternate embodiment is therefore a tradeoff of requiring fewer pipettings thus saving time vs. the added expense of using slightly more reagent. Furthermore, these larger wells may only be used when each slide is to be treated with the identical reagent. These larger wells are also useful in the cases for which large biological samples are examined with the single sample requiring several slides side by side for mounting. Trays with individual wells are suited to treating each slide with different reagents since each well is completely separate from nearby wells.
Another desirable feature which may be used with any embodiment of tray is to have trays of various colors which make it simple to determine which slides are undergoing which assay when a variety of assays is being performed.
A different embodiment of the invention is that rather than a tray, a special multichamber coverslip is placed on top of a slide which has a biological sample mounted on top of it. This special coverslip consists of three or six conjoined incubation chambers. A further feature of this special coverslip is that the top of each incubation chamber comprises a soft and pliable top rather than simply being a hard coverslip. The purpose of the soft top is to be able to push any trapped air bubbles to a region away from the biological sample. Another feature is that the special coverslip can include a raised region toward the edges of each chamber which can trap air which is pushed into the region and thus trap air bubbles which have been pushed to the edges thereby preventing the air bubbles from returning to the area of the slide on which the biological sample is mounted. Another advantage of the soft top is that the volume of reagent solution needed in a chamber becomes flexible.
An alternative embodiment of the special coverslip is to modify it to have tubing on one side of the chamber and a very small hole on the opposite side of the chamber. The tubing may contain a valve through which reagents can be added or removed and by which means the tubing can be closed. The small hole allows air to come out when reagent is added to the chamber. This modification allows the coverslip to be placed onto the slides prior to adding reagent, with reagent later being added via the tubing. Another desirable feature which may be used with any embodiment of coverslip is to have coverslips of various colors which make it simple to determine which slides are undergoing which assay when a variety of assays is being performed.
The third aspect of the invention is a set of slide covers which are premade as a set of multiple covers, preferably six covers, connected together and which may be laid on top of the processed slides which are still attached to the slideholder. The dimensions are such that all covers will perfectly line up on the set of slides. The covers are then easily detached from the holder. This may be accomplished by simply breaking them off by having a pre-scored region which allows for easily snapping off the coverslip from a xe2x80x9cholderxe2x80x9d region to which the slides are attached. The ability to simultaneously align and mount up to six slide covers is a time saving technique which is useful in such a labor intensive process.